Measuring the concentration of substances, particularly in the presence of other confounding substances and under varied conditions, is important in many fields such as medical diagnosis. For example, the measurement of glucose in body fluids, such as blood, is crucial to the effective treatment of diabetes.
Diabetic therapy typically involves two types of insulin treatment: basal and meal-time. Basal insulin treatment refers to continuous (e.g., time-released) insulin, often taken before bed. Meal-time insulin treatment provides additional dose boluses of faster-acting insulin to regulate fluctuations in blood glucose caused by a variety of factors, including the metabolization of sugars and carbohydrates. Proper regulation of blood glucose fluctuations requires accurate measurement of the concentration of glucose in the blood. Failure to do so can produce extreme complications, including blindness and loss of circulation in the extremities, which can ultimately deprive a diabetic of use of his or her fingers, hands, feet, etc.
Biosensor test strips often are used to measure the presence and/or concentrations of selected analytes in test samples. For example, a variety of test strips are used to measure glucose concentrations in blood to monitor the blood sugar level of diabetics. These test strips typically include a reaction chamber into which a reagent composition has been deposited. Current trends in test strips require smaller test samples and faster analysis times. This provides a significant benefit by allowing the use of smaller blood samples that can be obtained from less sensitive areas of the body, such as the forearm or the palm of the hand. Additionally, faster and more accurate test times provide better control of the diabetic's blood sugar level.
Multiple methods are known for measuring the concentration of analytes, such as glucose, in a blood sample. Such methods typically fall into one of two categories: optical methods and electrochemical methods. Optical methods generally involve reflectance or absorbance spectroscopy to observe a spectrum shift in a reagent. Such shifts are caused by a chemical reaction that produces a color change indicative of the concentration of the analyte. Electrochemical methods, however, generally involve amperometric, coulometric, potentiometric and/or conductive responses indicative of the concentration of the analyte. See, e.g., U.S. Pat. Nos. 4,233,029; 4,225,410; 4,323,536; 4,008,448; 4,654,197; 5,108,564; 5,120,420; 5,128,015; 5,243,516; 5,437,999; 5,288,636; 5,628,890; 5,682,884; 5,727,548; 5,997,817; 6,004,441; 4,919,770; 6,645,368; Re. 36,268 and 6,054,039.
In the consumer market segment, electrochemical methods typically use hand-held meters (but not always) to measure the electrochemical response of a blood sample in the presence of a reagent provided on a suitable biosensor. The reagent reacts with the glucose to produce charge carriers that are not otherwise present in the sample. Consequently, the electrochemical response of the blood is intended to be primarily dependent upon the concentration of blood glucose. Typical reagents used in electrochemical blood glucose meters and biosensors are disclosed in U.S. Pat. Nos. 5,997,817; 5,122,244; 5,286,362 and 7,727,467.
For example, biosensor test strips have been developed that employ the electrochemical principle of biamperometry. In one example, a biamperometric test strip contains the enzyme glucose dehydrogenase (GDH), which converts glucose in a blood sample to gluconolactone. This reaction liberates electrons that react with a mediator. In this example, an oxidized form of a mediator, such as hexacyanoferrate (III), accepts an electron and becomes a reduced form of the mediator, hexacyanoferrate (II). The meter applies a voltage between two electrodes, which causes the reduced mediator formed during a reaction incubation period to be reconverted to an oxidized mediator. This generates a small current that is read by the meter. One benefit is that biamperometric sensors do not require a true counter/reference electrode. Instead, the electrodes can be generally the same or substantially similar conductive materials, which in turn simplifies manufacturing of the test strip.
There are, however, a number of error sources that can create inaccurate results when measuring analyte levels in body fluid. For example, one issue with amperometric sensors is that the rate of reaction will affect the current response to a direct current (DC) potential measured at a predetermined time. Generally speaking, the reaction rate for a reactant or product in a particular reaction is traditionally defined as how fast a particular reaction takes place.
For amperometric tests, if the reaction rate is fast, then the current maximum and the measured current response may be higher than if the reaction rate is slow, even for sensors evaluating samples having the same concentration of analyte. For example, the reaction rate in an amperometric test is generally considered fast when the time to current maximum or peak current is less than that of other tests. Conversely, the reaction for a given amperometric test is typically considered slow when it takes longer to reach peak current in comparison to other tests. If the reaction rate is variable, then the current response at a given time will not be representative of an accurate measurement. As should be recognized, numerous factors can affect the reaction rate and its variability, such as temperature, diffusion rates and enzyme activity.
The effect of reaction rate is specifically important in the context of a fixed test time, such as those described herein, which are typical in systems used directly by consumers. For example, in the ACCU-CHEK® AVIVA® System employing a 5 to 6 second fixed test time, where the first 3 seconds constitute a fixed incubation time, the reaction is substantially complete by the time the measurement of glucose is made. In this system, an enzyme is employed such that the reaction is substantially complete under a wide variety of operating conditions such as temperature and aging of the test devices. As such, the enzyme is classified as “fast.” The rate of reaction problem can be exacerbated when an enzyme with a lower specific activity than the “fast” enzyme mentioned above and/or low reaction velocity is employed in the biosensor such as that used in embodiments herein. Under certain operating conditions, such as when the test is performed with an ambient temperature of 6° C., the reaction is not substantially complete until 10 or more seconds, which renders a fixed test time format of 5 seconds highly inaccurate. Sometimes harsh conditions to which the biosensor sensor is exposed worsen its accuracy.
Occasionally, the biosensor test strip can experience harmful conditions, often termed “strip rotting” or “vial abuse,” which refers to when the sensors are abused and exposed to detrimental conditions, such as excessive heat and/or moisture, during storage. This exposure to excessive heat and/or moisture also can result in slowing of the reaction times due to loss of enzyme activity leading to an inaccurate result using the fixed test time referred to above.
In the past, these issues have been avoided by using enzymes that have very fast reaction times and high specific activities with the analyte being measured. High loadings or amounts of enzymes in the biosensor also can help to avoid these reaction rate problems. By utilizing enzymes with these particular characteristics, reactions having high and similar levels of completion under all test conditions, such as at various temperatures and hematocrit levels, can be better achieved.
However, as a practical matter, some otherwise useful or desirable enzymes cannot be incorporated with high enough amounts into the biosensor without causing a significant loss in its performance. In addition, enzymes of high specific activities are not always desirable for all analytes. For example, many such systems are susceptible to adverse effects from various interfering substances (also referred to as non-analyte reacting compounds), such as maltose, galactose, xylose and the like, which can create inaccurate readings. Individuals undergoing peritoneal dialysis or Immunoglobulin G (IGG) therapy can experience high levels of maltose in their blood, which can interfere with accurate blood glucose readings. Therefore, interference from maltose can be a significant problem.
As an illustration, Abbott Laboratories' FREESTYLE® Blood Glucose Monitoring System employs a glucose-dye-oxidoreductase (GIucDOR) enzyme in conjunction with a coulometric technique with a variable test time to ensure robustness in view of varying degrees of reaction velocity. However, in addition to coulometry having a number of drawbacks making it impractical for many applications, such a system is still clinically unacceptable due to interference from maltose.
In addition to slowing enzyme activity, vial abuse also can result in an increase of background current, sometimes referred to as “blank current,” when readings are taken. There are a variety of sources for background or blank current. For instance, it is often desirable that mediators, which are used to transfer electrons from the enzyme to the electrode, be in an oxidized state before the biosensor is used. Over time, heat and/or humidity from vial abuse will tend to reduce the mediator. If part of the mediator is in a reduced form before the biosensor is used, a portion of the current will result from the working electrode oxidizing the reduced form of the mediator. The resulting background or blank current will tend to bias the signal, which in turn can lead to inaccurate results. Impurities in the reagent also can increase background or blank current problems.
Yet another source of error is blank current derived from other interfering compounds present in the blood as opposed to originating in the reagent. Examples of such error sources include ascorbate and acetaminophen, both of which are electrochemically active (or “electroactive”) and can react with either the electrode surface or the mediator used in the reagent layer. These and other compounds present in the fluid sample can contribute to a so-called blank current measured by the electrode that is not related to glucose concentration, and therefore provides a source of error.
Besides electroactive interfering compounds in the fluid sample itself, other interfering substances (such as non-analyte reacting compounds, the concentration of which may vary from sample to sample) can interact with various ingredients of the reagent and/or affect the manner in which the analyte diffuses. For example, GDH is an enzyme that catalyzes glucose, but also catalyzes other interfering substances, such as maltose, xylose, galactose and lactose. When the reagent used for the electrochemical testing includes GDH, the presence of one of these interfering sugars can adversely affect the measured glucose concentration. It also can be difficult to determine the concentrations of these interfering sugars prior to testing, and users who may have abnormal levels of such interfering sugars are frequently cautioned against using test strips with GDH. For example, disclaimers are used on many test strips with GDH reagents to assure that users with potential for abnormal levels of such interfering sugars do not test with GDH-based test strips.
As such, a need for reagent enzymes that are independent of those interfering sugars has been identified. However, some enzymes exhibiting this independence suffer from slow reactivity, which is a problem set forth and discussed above.
Interfering substances can occur naturally and can occur in varying concentrations in fluid samples. For example, the temperature of a fluid sample, or the concentration of red blood cells (hematocrit), uric acid, bilirubin or oxygen in body fluid can adversely influence the accuracy of blood glucose measurements.
Amperometric sensors have been proposed that use a “burn-off” approach to address at least some sources of the blank current problem. In this approach, two DC signals are applied to the sensor. The first DC signal, or burn-off signal, is used to consume or oxidize any species responsible for the blank current in the same diffusion layer adjacent to the working electrode that is later used to analyze the analyte. Afterwards, the second signal, or analysis signal, is used to analyze the analyte levels. Both the burn-off and analysis potentials have the same polarity and affect local concentrations of species at the same electrode and in a similar manner due to the common polarity. Although this burn-off technique reduces the effect of blank or background current, it does so at the expense of partially oxidizing (or reducing) the analyte to be measured, thereby reducing the signal-to-noise ratio of the sensor.
Algorithmic approaches in conjunction with the burn-off approach have been employed to define the relationship between burn-off current and measurement current produced by the respective two DC signals applied to the sensor. Nevertheless, such techniques have failed to compensate for variations in reaction time caused by factors such as enzymes with slow/variable reaction velocities. In addition, some enzymes used in such sensors, such as GDH, tend to be susceptible to maltose interference. Furthermore, such techniques require that the two signals are close enough in time such that additional reduced mediator does not diffuse back to the electrodes and effectively negate the reduction of blank and/or background current-causing species.
In contrast to GDH, glucose oxidase (GOx) exhibits a strong specificity for glucose and is generally maltose-independent. These features make GOx a suitable alternative to GDH when testing for glucose. However, GOx is an enzyme for which the natural terminal electron acceptor for glucose conversion is oxygen, and variations in the level of oxygenation of blood, such as is the case when comparing venous, arterial and capillary blood, can produce variations in the GOx response and can adversely affect the accuracy of glucose measurements using GOx.
A sharp relation between bias and blood oxygenation has been identified in GOx-based biosensors. As reflected in FIG. 1, the response bias (in milligrams per deciliter) varies considerably depending on the oxygenation of the sample (depicted in torr), and the measured glucose concentration can vary considerably depending on the oxygenation level of the sample when using GOx. See, e.g., FIGS. 2 and 3 depicting the test results of a biosensor using a GOx reagent that does not compensate for blood oxygenation. It was realized that test techniques are needed that accurately measure an analyte in a fluid, for example glucose in blood, in the presence of non-analyte reacting compounds.
For the foregoing reasons, it would be desirable for a sensor or measurement method to not be significantly affected by reaction time variability and to be insensitive to several sources of blank current. It also would be desirable if the sensor could be insensitive to both maltose levels and blood oxygen levels.